1. Field of the Invention
The present invention relates to naturally occurring truncated forms of Hepatocyte Growth Factor (HGF), which are encoded by alternative HGF mRNA transcripts. In particular, the present invention relates to a small HGF variant, HGF/NK2, which is a competitive antagonist of HGF, and HGF/NK1, which is a partial agonist of HGF.
2. Background Information
Hepatocyte growth factor has hormone-like activity and is released in response to partial hepatectomy and liver injury and is presumed to be an important mediator of liver regeneration (Nakamura et al., FEBS Lett. 224: 311 (1987); Gohda et al., J. Clin. Invest. 81: 414-419 (1988); R. Zarnegar and G. Michaelopoulous, Cancer Research 49: 3314-3320 (1989)). Its ubiquitous expression by stromal fibroblasts and demonstrated ability to stimulate DNA synthesis in melanocytes and endothelial cells as well as epithelial cells suggest that this factor plays a role in paracrine regulation of cell growth as well (Rubin et al., Proc. Natl. Acad. Sci. USA 88: 415 (1991)). Reports showing that scatter factor has high amino acid sequence identity to HGF over restricted regions, suggested that HGF may also be involved in modulating cell-cell interactions and migration (E. Gherardi and M. Stoker, Nature 346: 288 (1990); Weidner et al., J. Cell Biology 111: 2097-2108 (1990)). This has been borne out by subsequent studies that verify the identity of scatter factor and HGF.
Structurally, HGF resembles plasminogen in that it possesses characteristic kringle domains (Patthy et al., FEBS Lett. 171: 131-136 (1984)) and a serine protease-like domain (Miyazawa et al., Biochem. Biophys. Res. Commun., 163: 967-973 (1989); Nakamura et al., Nature 342: 440-443 (1989)). Like plasminogen, HGF can be processed by proteolytic cleavage, generating a heterodimeric molecule comprised of a heavy- and light-chain covalently linked by disulfide bonds (Nakamura et al., Nature 342: 440-443 (1989) and Miyazawa et al., Biochem. Biophys. Res. Commun. 163: 967-973 (1989)) The possibility that its actions might be mediated by a receptor tyrosine kinase was suggested by its rapid stimulation of tyrosine phosphorylation of cellular proteins in target cells (Rubin et al., Proc. Natl. Acad. Sci. U.S.A. 88: 415 (1991)). Recent studies have directly identified the HGF receptor as the c-met protooncogene product (Bottaro et al., Science 251: 802 (1991)), whose structure resembles that of a membrane-spanning tyrosine kinase (Park et al., Proc. Natl. Acad. Sci. USA 84: 6379-6383 (1987); Chan et al., Oncogene 2: 593-599 (1988)).
There is accumulating evidence that the positive effects of growth factors on cell proliferation can be counteracted at a variety of levels both intracellularly (Moses et al., Cell 63: 245-247 (1990) and at the cell surface (Hannum et al., Science 343: 336-340 (1990), Eisenberg, et al., Nature 343: 341-346 (1990); Carter et al., Nature 344: 633-637 (1990)). Thus, the potential exists to find an antagonist to HGF which would negatively regulate the growth factor""s proliferation effects. The invention described herein relates to small HGF variants and their corresponding transcripts. Characterization of one of these HGF variants, HGF/NK2, has revealed that it is a competitive antagonist of HGF action and thus establishes a novel regulatory mechanism whereby the same gene encodes both an agonist and antagonist of growth factor action. Characterization of another HGF variant, HGF/NK1, revealed that it is a partial agonist of HGF in vitro.
Although those of skill in the art of the invention possessed knowledge of HGF, no one knew about or could have predicted the existence of alternative mRNA transcripts encoding the claimed variants. Indeed, the inventors discovered HGF/NK1 and HGF/NK2, none of the work related to HGF even remotely suggested that a small transcript, particularly one, such as HGF/NK2, that is an HGF antagonist, or HGF/NK1, that is a partial HGF agonist, existed.
Thus, in one embodiment, the invention relates to a substantially pure HGF variant that is a truncated form of HGF comprising the N-terminal and first two kringle domains of HGF, and that specifically inhibits HGF-induced mitogenesis.
In another embodiment, the invention relates to a substantially pure HGF variant that is a truncated form of HGF comprising the N-terminal and the first kringle domain of HGF, and that is a partial HGF agonist.
In yet another embodiment, the invention relates to a method of inhibiting HGF induced mitogenesis in cells expressing the receptor for HGF, comprising contacting the cells with a mitogenesis-inhibiting amount of the HGF variant HGF/NK2, such that HGF induced mitogenesis is inhibited when the HGF variant binds the receptor for HGF on the cells.
In another embodiment, the invention relates to a method of stimulating mitogenesis in cells expressing the receptor for HGF, comprising contacting the cells with a mitogenesis-stimulating amount of HGF/NK1, such that mitogenesis is stimulated when the HGF variant binds the receptor for HGF on the cells.
In another embodiment, the invention relates to an isolated and substantially pure DNA molecule that encodes HGF/NK2.
In another embodiment, the invention relates to an isolated and substantially pure DNA molecule that encodes HGF/NK1.
In another embodiment, the invention relates to a recombinant vector comprising the above DNAs.
In another embodiment, the invention relates to a host cell stably or transiently transfected with the above described DNA in a manner allowing expression of the protein encoded by the DNA.
Another embodiment of the invention relates to a method of producing a recombinant HGF variant, comprising culturing the above host cell in a manner allowing expression of a protein and isolating the protein from the host cell.
In another embodiment, the invention relates to a method of producing the above described substantially pure HGF variant protein, the method comprising the following steps:
(i) culturing HGF variant-producing cells in a culture medium under conditions such that HGF variant is produced;
(ii) concentrating the culture medium so that a concentrate is formed;
(iii) contacting the concentrate with heparin under conditions such that HGF variant in the concentrate binds to the heparin, whereby a heparin-HGF variant complex is formed;
(iv) separating the heparin-HGF variant complex from the concentrate;
(v) treating the heparin-HGF variant complex under conditions such that the HGF variant dissociates from the heparin so that a solution of free HGF variant is formed;
(vi) fractionating the solution by sizing chromatography and/or reverse phase HPLC so that HGF variant is separated from the remaining components.
In another embodiment, the invention relates to a method of producing a substantially pure and biologically active Hepatocyte Growth Factor (HGF) variant comprising the steps of:
(i) disrupting HGF variant-producing bacteria that have been cultured in a culture medium under conditions such that HGF variant is expressed, so as to produce a first HGF variant protein-containing suspension;
(ii) recovering the protein from the first suspension and, washing and solubilizing the recovered protein, wherein the solubilizing is performed with a denaturant and reducing agent, and wherein a second protein-containing suspension is produced;
(iii) fractionating the second suspension by sizing chromatography with a solvent containing a denaturant and a reducing agent;
(iv) removing the denaturant from the fractions of step (iii) and pooling selected fractions containing denatured HGF variant;
(v) purifying said HGF variant in the pooled fractions by reverse phase chromatography;
(vi) lyophilizing the purified HGF variant of step (v) and redissolving the lyophilized HGF variant with denaturing and reducing agents;
(vii) serially diluting and then incubating the redissolved lyophilized proteins in refolding buffer, and then removing the denaturant by dialysis, so as to produce a third suspension containing biologically active proteins;
(viii) concentrating and then purifying said dialyzed proteins in the third suspension by sizing chromatography, so as to produce several fractions containing biologically active HGF variant; and
(ix) pooling and then concentrating the fractions containing biologically active HGF variant.
In a further embodiment, the present invention relates to therapeutic applications of the above described HGF variant that inhibits mitogenesis. Such applications could be used in proliferative disorders including both cancer and non-malignant conditions, in which HGF is excessive. The method comprises specifically blocking the action of HGF by administering a therapeutic amount of the HGF variant or by inducing the endogenous expression of increased amounts of the inhibitor.
The present invention also relates to therapeutic methods that decrease the overproduction of the HGF variant that inhibits mitogenesis. Such methods are applicable where HGF variant is inappropriately produced at high levels in a setting of impaired cell renewal. The method comprises specifically blocking the synthesis or action of the HGF variant by either contacting inhibitor HGF transcripts with antisense oligonucleotides or contacting inhibitor HGF protein with antibodies specific for the inhibitor molecules.
In yet another embodiment, the present invention relates to ex vivo methods of stimulating the growth of cells expressing the receptor for HGF. After proliferation, such cells can be transplanted into a subject in need of such cells. Such methods involve contacting the cells with growth-stimulating amounts of HGF/NK1 in culture prior to transplantation.
In yet another embodiment, the present invention relates to in vivo and in vitro methods of screening chemotherapeutic agents by using HGF/NK1 or HGF/NK2 as a carrier for chemotherapeutic agents that may be toxic to cells expressing the receptor for HGF. Because both HGF/NK1 and HGF/NK2 bind specifically to the HGF receptors on cells and are not themselves toxic, HGF/NK1 or HGF/NK2 can be used to carry potential toxins to such cells, thereby permitting evaluation of the efficacy of various toxins as chemotherapeutic agents.
In yet another embodiment, the invention relates to pharmaceutical compositions comprising either a mitogenesis-inhibitory amount of the HGF/NK2 or mitogenesis-stimulating amount of HGF/NK1 and a pharmaceutically acceptable carrier.